Metals are in widespread use to inhibit the growth of microbial populations. In particular, silver is commonly used in hospital settings as an effective broad spectrum biocide that has low toxicity to humans. However, protein systems are in place which enhance survival of microbes under conditions of high metal concentrations and reduce the efficacy of metals as broad spectrum biocides. One of these systems, the Cus system of E. coli, is responsible for sensing and responding to elevated levels of silver(I) and copper(I) in th environment. Despite its public health importance, the mechanisms of copper and silver handling by the Cus system are not well understood. We propose that mechanistic understandings of copper and silver handling will provide a target for future development of drugs and aid in understanding the limitations of metal-based biocides. In our previous work, we have focused on using the Cus system as a model system for a large class of microbial metal resistance systems. We have biochemically and structurally characterized two of the components, CusF and CusB, of the membrane spanning metal efflux system (CusCFBA) that actively removes metals from the cell. Additionally we have developed a novel and powerful technique to monitor metal transfer between proteins. The natural development of this previous work is to integrate these findings with the other proteins in the system. In aim 1 we will investigate the mechanism of metal transport by the Cus system and test the hypothesis that the Cus system is primarily important for detoxification of the periplasm, through metal transfer from the Cus periplasmic proteins (CusB and CusF) to the membrane-bound Cus proteins (CusA and CusC). In aim 2, we will test the hypothesis that CusS is activated by metal binding to the periplasmic domain, leading to conformational changes that ultimately result in activation of Cu(I)/Ag(I) resistance systems.. At the conclusion of these studies, we will have a detailed understanding of the microbial counterattack to metal contaminated environments. We will have determined how a specific metal is discriminated from other metals by a sensory and response system, the mechanisms of the transport process, and the further detoxification strategy after transport has occurred. These studies will give us the tools to understand the microbial response to metal biocides and provide strategies for their effective use in controlling bacterial infections in human populations.